Polypeptide for detecting ACPA negative rheumatoid arthritis diagnostic marker and application thereof

Abstract

The invention provides a polypeptide for detecting an ACPA negative rheumatoid arthritis diagnosis marker and application of the polypeptide. The rheumatoid arthritis diagnostic marker is an anti-P antigen family protein 5 antibody, and the amino acid sequence of the polypeptide of the antibody, namely the P antigen family protein 5, is as shown in SEQ ID NO. 1. The invention also discloses application of the P antigen family protein 5 in preparation of a reagent for diagnosing ACPA-negative rheumatoid arthritis. By detecting the rheumatoid arthritis diagnostic marker, the kit can be effectively used for detecting the rheumatoid arthritis, the detection rate is increased, and a basis is provided for preventing and treating the rheumatoid arthritis as soon as possible.

Description

Technical Field

[0001] The invention belongs to the field of biological detection, and specifically relates to a polypeptide for detecting an ACPA-negative rheumatoid arthritis diagnostic marker and an application thereof. Background Art

[0002] Rheumatoid arthritis (RA) is a chronic autoimmune disease that mainly causes inflammatory reactions in the synovium of multiple small joints throughout the body, which in turn causes local bone destruction in the joints. However, the sensitivity of existing RA diagnosis is not high, and a considerable number of early RA are not identified. But at the same time, it is particularly important that new RA cases may benefit from early effective intervention to avoid progression to chronic, erosive RA, further leading to disability, affecting long-term quality of life, and increasing disease mortality. Therefore, for rheumatoid arthritis, early diagnosis and treatment are the key to preventing irreversible joint destruction. In the latest RA classification criteria in 2009, serum ACPA (anti-citrullinated polypeptide antibody) positivity was included in the diagnostic criteria. This anti-citrullinated protein antibody is considered to be a serum-specific marker for rheumatoid arthritis. At the same time, there is also a class of RA patients with negative ACPA antibodies in clinical practice. With the deepening of understanding of the disease, it is gradually recognized that there is a certain clinical heterogeneity between RA patients with positive ACPA antibodies and RA patients with negative ACPA antibodies. However, the lack of specific biomarkers for RA patients with negative ACPA antibodies in clinical practice has greatly increased the difficulty of diagnosing such patients. There are also reports of new autoantibody subtypes in the serum of RA patients that recognize carbamylated proteins (anti-CarP). This antibody system is independent of ACPA, because the serum antibodies of RA patients can distinguish between citrullinated and carbamylated antigens. Accordingly, a considerable number of ACPA-negative patients have anti-CarP antibodies. In the past few years, due to the discovery of RA-specific autoantibodies against citrullinated and carbamylated proteins, people have a deeper understanding of the pathogenesis and etiology of RA. Carbamylation and citrullination are both post-translational modifications that cause protein carbamylation and citrullination, respectively, replacing positively charged amino acids with neutral amino acids. Citrullinated proteins are produced by the PAD enzyme, while carbamylated proteins are converted to homocitrulline by chemical reactions from the amino acid lysine. Citrulline and homocitrulline are similar in chemical structure, but are located at different sites in the protein, because arginine and lysine are in different sites. Moreover, homocitrulline only has an additional formyl group. Although some antibodies are reactive to both structures, some anti-CarP antibodies do not react to citrulline, and similarly, some anti-citrulline antibodies do not react to CarP.

[0003] RA is a chronic autoimmune disease, and the detection of autoantibody markers plays an important role in the diagnosis of the disease. It is very important to correctly identify RA in the early stage. Identifying more ACPA-negative RA-related autoantibody markers and some autoantigens that can predict disease activity and efficacy is very important for reducing the disability rate of RA. It is very important to determine the diagnostic markers of ACPA-negative rheumatoid arthritis and improve its detection rate. Summary of the invention

[0004] In order to solve the above technical problems, the present invention provides polypeptides for detecting ACPA-negative rheumatoid arthritis diagnostic markers and applications thereof.

[0005] To achieve the above object, the present invention adopts the following technical solutions:

[0006] The ACPA-negative rheumatoid arthritis diagnostic marker provided by the present invention is an anti-P antigen family protein 5 (Pantigen family member 5, PAGE5) antibody.

[0007] The present invention also provides the use of the polypeptide in preparing a reagent for diagnosing ACPA-negative rheumatoid arthritis. Furthermore, the amino acid sequence of the polypeptide, i.e., P antigen family protein 5, is shown in SEQ ID NO.1. Furthermore, the gene sequence encoding P antigen family protein 5 is shown in SEQ ID NO.2.

[0008] Wherein, the diagnosis comprises: determining the level of antibodies against P antigen family protein 5 in a biological sample obtained from a patient with ACPA-negative rheumatoid arthritis; optionally,

[0009] The level of antibodies in the biological sample is compared to control data, wherein detectability of antibodies reactive to PAGE5 in the sample relative to the control data indicates a likelihood of having ACPA-negative rheumatoid arthritis.

[0010] Wherein, the biological sample is a serum sample.

[0011] The level of PAGE5 antibody is measured by the following steps:

[0012] S1, contacting a serum sample from a patient with P antigen family protein 5;

[0013] S2, formation of antibody-protein complex between antibodies present in serum samples and P antigen family protein 5;

[0014] S3, washing to remove any unbound antibody;

[0015] S4, adding a detection antibody that is labeled and reactive to the antibody from the serum sample;

[0016] S5, washing to remove any unbound labeled detection antibody; and

[0017] S6. Converting the marker of the detection antibody into a detectable signal; wherein the presence of the detectable signal indicates the presence of anti-P antigen family protein 5 antibodies in the patient.

[0018] Wherein, the P antigen family protein 5 is deposited or fixed on a solid support.

[0019] Wherein, the support is fluorescent microspheres, porous plates or colloidal gold.

[0020] Wherein, the detection antibody is labeled by covalently linking to an enzyme, a marker having a fluorescent compound or a metal, or a marker having a chemiluminescent compound.

[0021] Furthermore, the detection antibody is an anti-human IgG antibody labeled with PE-Cy5.

[0022] The beneficial effects of the present invention are:

[0023] The polypeptide for detecting the diagnostic marker of ACPA-negative rheumatoid arthritis and its application provided by the present invention can greatly improve the detection rate of ACPA-negative rheumatoid arthritis by using the polypeptide to detect the anti-PEGE5 antibody in the serum. Through the detection of protein chip, the risk of RA can be successfully predicted and prevented in advance. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 This is the detection result of the protein chip coated with protein 1 in Example 3.

[0025] Figure 2 This is the detection result of the protein chip coated with protein 2 in Example 3. DETAILED DESCRIPTION

[0026] The following examples are used to further illustrate the present invention, but should not be construed as limiting the present invention. Without departing from the spirit and substance of the present invention, modifications or substitutions made to the present invention all belong to the scope of the present invention.

[0027] Unless otherwise specified, the technical means used in the examples are conventional means well known to those skilled in the art. Unless otherwise specified, the reagents used in the present invention are all analytically pure or above.

[0028] The samples used in the present invention are 555 serum samples provided by the rheumatology and immunology department of a medical institution, including: 298 sera from patients diagnosed with RA, with an average age ± standard deviation of 45.2 ± 12.5; 48 sera from systemic lupus erythematosus, with an average age ± standard deviation of 36.8 ± 12.4; 28 sera from Behcet's disease, with an average age ± standard deviation of 54.2 ± 20.7; 132 sera from healthy people, with an average age ± standard deviation of 37.5 ± 12.1; and 10 sera from Takayasu arteritis, with an average age ± standard deviation of 38.4 ± 13.5; wherein the diagnosis of RA is in accordance with the standards established by ACR / EULAR in 2010, and other patients also meet their respective diagnosis and / or classification standards.

[0029] All sera were collected by the Department of Rheumatology and Immunology of medical institutions from 2006 to 2014. All disease sera were from clinically diagnosed patients. Patients with controversial diagnoses had their diagnoses confirmed after consultation with at least three chief physicians. All RA sera were tested for serum corresponding antibodies, including ANA 3 items: DNA-IF (immunofluorescence), ANA-IF (immunofluorescence) and ds-DNA (ELISA), anti-CCP antibody, i.e. ACPA test (>25IU / ml defined as positive), RF test, AKA and APF test, MCV test, GPI test. All RA patients with negative anti-CCP antibodies and / or anti-AKA, APF, MCV antibodies met the diagnostic criteria for RA synovitis by ultrasound or MRI.

[0030] Example 1 Selection of PAGE5 antigen protein

[0031] 1.1 Construction of recombinant plasmid

[0032] P antigen family protein 5 protein (PAGE5) was selected, and its nucleotide sequence can be found in GNnBank sequence number: NM_130467.5. The gene sequence was modified according to the preferred codons of Escherichia coli and the original protein was truncated. After a large number of experimental studies, an increased expression efficiency was obtained, and the expression efficiency of foreign proteins reached more than 70%. The secondary structure of the recombinant protein was screened by bioinformatics. Two optimized gene sequences are now cited for illustration. The gene sequences are shown in SEQ ID NO.2 and SEQ ID NO.3, and the corresponding amino acid sequences are shown in SEQ ID NO.1 and SEQ ID NO.4. Shanghai Shenggong Company was commissioned to synthesize the gene sequences (SEQ ID NO.2, SEQ ID NO.3), and the genes were cloned into the expression vector pNT30a to obtain recombinant plasmids 1 and 2.

[0033] SEQ ID NO.1: GNRGWAGTREEVRDMSEHVTRSQSSERGNDQESSQP VGPVIVQQPTEEKRQEEEPPTDNQGIAPSGEIKNEGAPAVQGTDVEAF

[0034] SEQ ID NO.2: GGCAATAGAGGCTGGGCAGGCACAAGAGAAGAAG TTAGAGATATGTCAGAACATGTTACAAGATCACAATCATCAGAAAGAGGCAATGATCAAGAATCATCACAACCGGTTGGCCCGGTTATTGTTCAACAACCGACAGAAGAAAAAAGACAAGAAGAAGAACCGCCGACAGATAATCAAGGCATTGCACCGTCAGGCGAAATTAAAAATGAAGGCGCACCGGCAGTTCAAGGCACAGATGTTGAAGCATTT;

[0035] SEQ ID NO.3: ACCCGCGAAGAAGTGCGCGATATGAGCGAACATG TGACCCGCAGCCAGAGCAGCGAACGCGGCAACGATCAGGAAAGCAGCCAGCCGGTGGGCCCGGTGATTGTGCAGCAGCCGACCGAAGAAAAACGCCAGGAAGAAGAACCGCCGACCGATAACCAGGGCATTGCGCCGAGCGGCGAAATTAAAAACGAAGGCGCGCCGGCGGTGCAGGGCACCGATGTGGAAGCGTTTCAGCAGGAACTGGCGCTGCTGAAAATTGAAGATGCGCCGGGCGAT;

[0036] SEQ ID NO.4: TREEVRDMSEHVTRSQSSERGNDQESSQPVGPVIVQ QPTEEKRQEEEPPTDNQGIAPSGEIKNEGAPAVQGTDVEAFQQELALLKIE DAPGD。

[0037] 1.2 Expression and identification of recombinant protein

[0038] The recombinant plasmids 1 and 2 with correct sequencing were transformed into Escherichia coli BL21, and the single colony with correct identification was inoculated into LB liquid culture medium containing ampicillin (concentration of 10 μg / mL), and cultured at 37°C overnight with shaking. The next day, it was inoculated into a new LB liquid culture medium containing ampicillin at a ratio of 1:50 and cultured with shaking until OD600 reached 0.7, then the bacterial solution temperature was cooled to 22°C, IPTG was added to make its final concentration 0.75 mmol / L, and induced expression was performed at 22°C; 1 mL of bacterial solution was drawn after 6 hours, and lysed by ultrasonication for 10 minutes, the bacterial solution was collected, boiled in a water bath for 10 minutes, centrifuged at 4°C and 10000 r / min for 5 minutes, placed on ice, and the supernatant and precipitate were drawn for 12% SDS-PAGE electrophoresis detection; after the electrophoresis, the gel was stained with Coomassie Brilliant Blue for 90 minutes, and the protein induced expression was observed after decolorization for 2 hours, and the target protein appeared in the precipitate, which was about 10KD and 11KD in size.

[0039] 1.3 Purification of recombinant protein

[0040] 2 mL of induced expression bacterial solution was taken separately, centrifuged at 4°C, 12000 r / min for 30 minutes, the cell pellets were collected, washed three times with 0.01M pH7.4 PBS, suspended in pH8.0 lysis buffer (Tris-Nacl), ultrasonicated for 15 minutes, centrifuged at 4°C, 12000 r / min for 30 minutes, the pellets were suspended in 8 mol / L urea at pH8.0, shaken on ice for 2 hours to dissolve inclusion bodies, centrifuged at 12000 r / min for 30 minutes, the supernatant was filtered through a 0.22 μm filter membrane and purified using a His Trap TMHP (histidine) affinity adsorption column purification system, the purity was identified by SDS-PAGE, and finally the purified protein was dialyzed into 0.01 mol / L, pH7.4 PBS, concentrated by PNG-20000, and the target protein molecular weights of approximately 10KD and 11KD were obtained. Purified recombinant protein 1 and protein 2.

[0041] Example 2 Large-scale preparation of P antigen family protein 5 protein

[0042] Regarding the selection of P antigen family protein 5 protein, the inventors of the present invention conducted multiple experiments and finally determined to select P antigen family protein 5 protein, whose amino acid sequence is shown in SEQ ID NO.1. The gene sequence was modified according to the preferred codons of Escherichia coli, and the secondary structure of the recombinant protein was screened by bioinformatics. After multiple experimental verifications, the final gene sequence was determined as shown in SEQ ID NO.2.

[0043] The following examples illustrate the experiment with two gene sequences and amino acid sequences. The sequences shown in SEQ ID NO.2 and SEQ ID NO.3 were commissioned to be synthesized by Shanghai Yingjun Company and connected to the expression vector pNT30a respectively to obtain the connection product, i.e., the recombinant plasmid, which was transformed into DH5α competent cells. The recombinant plasmid was identified by PCR and double enzyme digestion, and the positive clones were screened and sent to Shanghai Jun Company for sequencing. The correctly sequenced recombinant plasmid was transformed into Escherichia coli BL21, and the correctly identified single colony was inoculated into LB liquid culture medium containing ampicillin, and cultured at 37°C overnight with shaking. The next day, it was inoculated into a new LB liquid culture medium containing ampicillin at a ratio of 1:100 and cultured with shaking until OD600 reached 0.8, and then cooled. The bacterial solution temperature was raised to 22°C, and IPTG with a final concentration of 0.8 mmol / L was added to induce expression at 22°C; 1 mL of bacterial solution was drawn at 1h, 2h, 3h, 4h, 5h, and 6h, respectively, and ultrasonically lysed, the bacterial solution was collected, boiled at 100°C for 10min, centrifuged at 4°C and 12000r / min for 3min, placed on ice, and the supernatant was drawn for SDS-PAGE electrophoresis detection; after the electrophoresis, the gel was stained with Coomassie Brilliant Blue for 80min, and then decolorized for 2h to observe the protein induced expression; the target protein was expressed from 1h after induction, and the yield was highest at 6h.

[0044] 2 ml of bacterial solution induced for 6 hours of expression was taken and centrifuged at 12000 r / min for 30 minutes, the cell pellet was collected, washed three times with 0.01 M pH 7.4 PBS, suspended in pH 8.0 lysis buffer (Tris-NaCl), sonicated, and centrifuged. 10 μl of supernatant and precipitate were taken for SDS-PAGE, and the gel was stained with Coomassie Brilliant Blue R-250 after electrophoresis. The specific target protein band was checked after decolorization with decolorizing solution. The results showed that the recombinant protein was expressed in the form of inclusion bodies. The cell precipitates (mainly containing some cell debris and inclusion bodies) were collected after ultrasonic centrifugation, suspended in 8 mol / L urea at pH 8.0, and shaken on ice for 2 hours to dissolve the inclusion bodies. The supernatant was centrifuged at 12000r / min for 30 minutes, and the supernatant was filtered through a 0.22μm filter membrane and purified using a His Trap TMHP (histidine) affinity adsorption column purification system. The purity was identified by SDS-PAGE. Finally, the purified protein was dialyzed into 0.03mol / L, pH 7.4 PBS and concentrated with PEG-20000 to obtain purified recombinant proteins with molecular weights of approximately 10KD and 11KD.

[0045] The N-termini of the obtained recombinant protein 1 (the corresponding amino acid sequence is shown in SEQ ID NO.4, denoted as protein 1) and recombinant protein 2 (the corresponding amino acid sequence is shown in SEQ ID NO.1, denoted as protein 2) of P antigen family protein 5 have glutathione S-transferase (GST) tags. The recombinant protein of P antigen family protein 5 obtained above is coated on the chip by conventional methods. The chip quality is verified by hybridization with mouse anti-GST monoclonal antibody to the chip. When the correlation of the fluorescence signal values ​​of every two repeated protein spots on the chip reaches more than 97%, it is considered that the repeatability of every two spots on the chip is good. 238 RA sera and 197 control sera (38 SLE sera, 9 Takayasu arteritis sera, 18 Behcet's disease sera and 132 healthy human sera) were selected for hybridization with protein chips, so that antibody-protein complexes were formed between the antibodies present in the serum samples and PAGE5; then washed to remove any unbound antibodies; anti-human IgG antibodies labeled with PE-Cy5 were added for reaction, and after the reaction, the sera were detected with anti-human IgG antibodies labeled with PE-Cy5, and identified by signal acquisition and data analysis. The reaction of P antigen family protein 5 protein antibodies in serum with P antigen family protein 5 protein antigen probes was detected with anti-human IgG antibodies labeled with PE-Cy5.

[0046] Specifically, the antibody level of PAGE5 is measured by the following steps, including:

[0047] S1. contacting a serum sample from a patient with a recombinant protein of P antigen family protein 5;

[0048] S2, formation of antibody-protein complex between antibodies present in serum samples and P antigen family protein 5;

[0049] S3, washing to remove any unbound antibody;

[0050] S4, adding a labeled detection antibody reactive to the antibody from the serum sample, i.e., a PE-Cy5 labeled anti-human IgG antibody;

[0051] S5, washing to remove any unbound labeled detection antibody; and

[0052] S6. Converting the marker of the detection antibody into a detectable signal; wherein the presence of the detectable signal indicates the presence of anti-P antigen family protein 5 antibodies in the patient.

[0053] Scan and obtain the fluorescence signal graph of each chip, and drag the graph and the template file of the chip, i.e., the gail file, into the GenePix Pro 6.0 software for one-to-one correspondence. Then convert the signal information of all probes on each chip collected by the GenePix Pro 6.0 software and import it into an Excel table. The foreground signal intensity (F635median) of each probe point minus the surrounding background signal intensity (B635median) is used as the signal value of the point.

[0054] First, the data of ACPA-negative RA patients and healthy controls and disease controls were subjected to chi-square test or Fisher's exact test. As diagnostic marker proteins, parameters such as T score and p value can be obtained; for each protein, 1000 different cutoff values ​​were selected, and sensitivity and specificity can be calculated according to each cutoff value. The ROC curve was drawn with these 1000 points (1-specificity, sensitivity), and the AUC was calculated. The cutoff value corresponding to the point with the highest sum of sensitivity and specificity was the optimal cutoff. The results are shown in Table 1. In the results of hybridization reaction with large sample serum, the sensitivity of protein 2 antigen in immune reaction with ACPA-negative RA serum was greater than 25%, and it also had specificity different from healthy controls and disease controls. Protein 1, cutoff value was 2.0840, and its sensitivity was 33.60%, and protein 2, cutoff value was 2.0683, and its sensitivity was 66.80%.

[0055] Table 1 Cutoff values ​​and corresponding AUCs of protein 1 and protein 2 in different ACPA-negative RA

[0056] cutoff value P-value Sensitivity Specificity AUC Protein 1 2.0840 0.000 0.3360 0.9590 0.695 Protein 2 2.0683 0.000 0.6680 0.9090 0.8650

[0057] Example 3

[0058] External serum samples (60 RA sera, 10 SLE sera, 10 Takayasu arteritis sera, 10 Behcet's disease sera, and 30 healthy human sera) were used to verify the obtained protein 1 and protein 2. The protein chip prepared with protein 1 and protein 2 in Example 2 was used for detection to obtain the fluorescence value. The results were judged based on the cutoff value. Figure 1 and Figure 2 The accuracy of protein 1 and protein 2 obtained in the test results is 0.6250, and the accuracy of protein 2 is 0.8000, which is higher and can avoid missed detection. Therefore, protein 2 obtained from the SEQ ID NO.2 gene sequence can be used as a diagnostic marker for ACPA-negative rheumatoid arthritis and can be used for the detection of ACPA-negative rheumatoid arthritis.

[0059] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the technical principles of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A polypeptide for detecting a diagnostic marker for ACPA-negative rheumatoid arthritis, characterized in that: The diagnostic marker is an anti-P antigen family protein 5 antibody, and the polypeptide is P antigen family protein 5, and its amino acid sequence is shown in SEQ ID NO.

1.

2. The gene sequence encoding the polypeptide according to claim 1 is shown in SEQ ID NO.

2.

3. Use of the polypeptide according to claim 1 in the preparation of a reagent for diagnosing ACPA-negative rheumatoid arthritis.

4. The use according to claim 3, characterized in that: The risk of rheumatoid arthritis in patients who present with ACPA negativity is determined by measuring the level of antibodies against P antigen family protein 5 in biological samples.

5. The use according to claim 4, characterized in that: Detecting the level of antibodies corresponding to P antigen family protein 5 in a biological sample comprises the following steps: S1, contacting a serum sample from a patient with P antigen family protein 5; S2, formation of antibody-protein complex between antibodies present in serum samples and P antigen family protein 5; S3, washing to remove any unbound antibody; S4, adding a detection antibody that is labeled and reactive to the antibody from the serum sample; S5, washing to remove any unbound labeled detection antibody; S6. Converting the marker of the detection antibody into a detectable signal; wherein the presence of the detectable signal indicates the presence of anti-P antigen family protein 5 antibodies in the patient.

6. The use according to claim 5, characterized in that: The P antigen family protein 5 is deposited or fixed on a solid support.

7. The use according to claim 5, characterized in that: The support is fluorescent microspheres, a porous plate or colloidal gold.

8. The use according to claim 5, characterized in that: The detection antibody is labeled by covalently linking to an enzyme, a label with a fluorescent compound or metal, or a label with a chemiluminescent compound.

9. The use according to claim 5, characterized in that: The detection antibody is an anti-human IgG antibody labeled with PE-Cy5.

Images